Bridging pathways: SBP15 regulates GOBLET in modulating tomato axillary bud outgrowth

This article comments on: Barrera-Rojas CH, Vicente MH, Brito DAP, Silva EM,Muñoz Lopez A, Ferigolo LF, Carmo RM, Silva CMS, Silva GFF, Correa JPO, Notini MM, Freschi L, Cubas P, Nogueira FTS. 2023. Tomato miR156-targeted SlSBP15 represses shoot branching by modulating hormone dynamics and interacting with GOBLET and BRANCHED1b. Journal of Experimental Botany 74, 5124–5139.

The influence of the shoot apex on suppressing the growth of lower organs is well known.In orchards, gardeners prune the tips of plants to promote growth in lower areas.However, in plants such as tomatoes, excessive shoot branching diverts resources from fruit development and reduces yield (Pino et al., 2022).Consequently, it is desirable to eliminate or reduce shoot branching in tomatoes to avoid manual pruning of axillary branches.Mutations in tomato, such as LS (Schumacher et al., 1999) and BL (Schmitz et al., 2002) suppress branching but also negatively affect fruit yield (Groot et al., 1994).
Our knowledge of the mechanisms governing AB formation, quiescence, and outgrowth through the activation of axillary meristems (AMs) is limited.Auxin, a plant hormone, plays a crucial role in regulating AB outgrowth.Application of auxin to a decapitated shoot apex restores apical dominance and inhibits AM activation, establishing auxin as a master regulator of AB growth.Although auxin is transported basipetally in the stem, it does not enter ABs.Inhibition of basipetal auxin transport in the stem by phytotropic agents promotes AB outgrowth (Beveridge et al., 2000;Fichtner et al., 2017).While auxin acts as an inter-organ communicator, a specific gene module regulates the suppression or activation of AB growth.Transcription factors belonging to the TCP family, such as TEOSINTE BRANCHED1 (TB1) or BRANCHED1 (BRC1), negatively regulate AB outgrowth (Liu et al., 2017).In tomato, two paralogs of BRC1, BRC1a and BRC1b, are expressed in dormant ABs and down-regulated upon bud activation.Loss-of-function studies indicate that BRC1b plays a role similar to the ancestral BRC1 gene in suppressing AB growth (Martín-Trillo et al., 2011).
The expression levels of BRC1 genes are regulated by the SPL/SBP transcription factor family, which is modulated by miR156 (Xie et al., 2020).The miR156/SBP/SPL regulatory hub operates through the cleavage or translational repression of SPL transcripts by miR156 (Wang and Wang, 2015).Overexpression of miR156 in tomato leads to increased shoot branching (Silva et al., 2014) and a reduction in SPL/SBP13 transcripts (Cui et al., 2020).While the miR156/SBP/SPL module regulates shoot branching in tomato, the downstream processes are still not fully understood.

miR156-resistant SBP15 elicits strong quiescence in axillary buds
To investigate the molecular basis of shoot branching, Barrera-Rojas et al. (2023) manipulated genetic pathways associated with AM activation in Micro-Tom, a dwarf tomato variety.Consistent with previous reports, Micro-Tom plants miR156 displayed more activated ABs than wild-type plants.This phenotype was not limited to Micro-Tom, as the high shoot branching trait could be transferred to a commercial tomato cultivar-Moneymaker.These results established that miR156-targeted genes negatively regulate shoot branching in tomato.
One target of miR156 is the SPL/SBP gene family, whose members act as repressors of shoot branching in other species such as rice (Dai et al., 2018) and Arabidopsis (Gao et al., 2018).Barrera-Rojas et al. generated a phylogenetic tree comparing SBP-box domains from different species to identify potential targets of miR156 in regulating tomato branching.Among the 15 SPL/SBP genes in tomato, SBP15 grouped with known shoot branching-associated SPL genes.This homology suggested that SBP15 could be the target of miR156 for negatively regulating shoot branching in tomato.The expression pattern of SBP15 transcripts overlapped with the miR156 expression domain in ABs, further supporting this notion.
To investigate the role of SBP15 in regulating ABs, Barrera-Rojas et al. overexpressed an miR156-resistant version of SBP15 (rSBP15) in Micro-Tom.The rSBP15 plants exhibited AB formation, but the buds were smaller and remained at an early/dormant stage.Surprisingly, a gene-edited null mutant of SBP15 (sbp15 CRISPR ) did not show noticeable changes in shoot branching compared with wild-type plants, indicating that the down-regulation of SBP15 by miR156 was sufficient to initiate ABs in tomato.The phenotypes of rSBP15 and sbp-15 CRISPR plants collectively indicated that the miR156-targeted down-regulation of SBP15 initiates AB outgrowth in tomato, possibly in conjunction with other SBPs such as SBP13 (Cui et al., 2020), to establish the vegetative architecture of tomato.

Strong axillary bud quiescence was associated with elevated auxin/abscisic acid levels
The smaller and less developed ABs in rSBP15 plants compared with those of Micro-Tom suggested that the miR156/ SBP15 pathway might affect the polar auxin transport stream (PATS).By hindering auxin export from ABs, rSBP15 probably restricted their outgrowth.This view was supported by the decapitation experiment, where the topmost AB in rSBP15 plants exhibited stronger dominance and grew significantly longer than the lower ABs.Additionally, the application of a PATS inhibitor [1-naphthylphthalamic acid (NPA)] revealed that rSBP15 ABs were more responsive to the reduction in auxin transport compared with Micro-Tom ABs.These findings suggest that miR156-targeted SlSBP15 may influence auxin transport in ABs, affecting their activation and growth.
The regulation of auxin synthesis and transport is complex, and its relationship with the miR156/SBP15 pathway is not well understood.To unravel the underlying genic regulation causing AB arrest, Barrera-Rojas et al. analyzed the transcriptome of ABs from Micro-Tom and its transgenic 156-OE and rSBP15 plants.Consistent with the quiescence of ABs in rSBP15 plants, genes related to cell division and protein synthesis were transcriptionally repressed.The transcriptome analysis also revealed that a marker for AB dormancy, DRM1, was down-regulated in 156-OE plants and up-regulated in rSBP15 ABs.Notably, auxin-associated genes, including LAX2 and PIN9 involved in auxin transport, were down-regulated in rSBP15 ABs, correlating with their growth arrest.The rSBP15 ABs had higher endogenous indole-3-acetic acid (IAA) levels and accumulated abscisic acid (ABA), in line with their arrested development.An up-regulation of ABA is also observed in arrested ABs of shaded Arabidopsis plants wherein BRC1 activates genes encoding HD-ZIP transcription factors, which trigger a genetic cascade elevating ABA levels by up-regulating NCED, an enzyme involved in ABA biosynthesis (González-Grandío et al., 2017).

SBP15 suppresses GOBLET expression in quiescent buds
In species such as wheat, rice, and Arabidopsis, homologs of SBP15 genes inhibit shoot branching by activating TB1/ BRC1-like genes.In tomato, BRC1a and BRC1b are expressed in arrested ABs and down-regulated upon bud activation.Consistent with previous reports that BRC1b is the primary regulator of branching (Martín-Trillo et al., 2011), yeast twohybrid (Y2H) assays and intensity-based Förster resonance energy transfer (APB-FRET) assays demonstrated that SBP15 interacts with BRC1b but not with BRC1a.Functional analysis revealed that SBP15 and BRC1b cooperatively enhanced the activity of the NCED promoter but had opposite effects on the PIN9 promoter.A direct interaction between SBP15 and BRC1b proteins in tomato is surprising, as in rice, the SBP15 homolog IPA1/SPL14 is an upstream regulator of OsTB1 expression (Lu et al., 2013).Nonetheless, the above findings suggest that SlSBP15 and SlBRC1b can modulate the expression of common targets in a cooperative or antagonistic manner.
An intriguing discovery was made regarding the down-regulation of the miR164-targeted gene GOBLET (GOB) in the ABs of both 156-OE and rSBP15 plants (Fig. 1).In tomato, GOB plays a prominent role in leaf dissection, and its expression is regulated by auxin.The significant reduction in GOB transcript levels in rSBP15 ABs suggested that SBP15 might directly target GOB.Co-expression analysis in Nicotiana benthamiana leaves confirmed that high levels of rSBP15 repressed the expression of GOB by probably binding to its promoter.Crossing rSBP15 plants with a Gob-4d mutant only partially restored the highly branched phenotype of the mutant, indicating that rSBP15 had epistatic control over the highly branched Gob-4d mutant.These findings establish the reduction of GOB as a novel player in regulating AB outgrowth.
The analysis conducted by Barrera-Rojas et al. expands our understanding of the partners involved in the miR156/SBP-mediated shoot branching regulation in tomato.study highlights the interconnectedness of the tomato miR156/SBP15 module with auxin, ABA, GOB, and the expected SlBRC1b gene expression.All these players contribute to the regulatory circuit controlling tomato's vegetative architecture.The study suggests that maintaining low levels of GOB transcripts is crucial for dormant ABs with high SBP15 expression.However, the SBP15-dependent repression of GOB is complex and probably occurs through both auxin-dependent and independent pathways within ABs.
Although the growth of ABs with high SBP15 expression is impaired, they can still respond to the interruption of polar auxin transport (PAT).This suggests that the development of auxin canalization, which involves specific cell files differentiating into vascular tissues, may be insufficient in smaller ABs with high SBP15 expression.However, these ABs can grow when the supply of auxin from the shoot apex is disrupted by decapitation.

What lies in the future?
It is worth noting that several hypotheses have been proposed to explain the release of ABs after decapitation.The prominent one is the 'auxin canalization' hypothesis, which states that ABs can only grow once auxin export is initiated.The PIN1 efflux carrier dominantly mediates PAT.It is plausible that PIN9 initiates canalization during early AB activation, followed by the takeover by PIN1.However, the precise roles of different PIN proteins in the onset of auxin canalization await further investigation through in situ expression analysis during AB activation.It is worth mentioning that auxin transport can be influenced by various factors.For example, in tomato, the outgrowth of ABs is also a result of the down-regulation of cytokinin levels in plants that overexpress cytokinin oxidase (Pino et al., 2022).
Although auxin plays a central role in all hypotheses related to the onset of AB outgrowth, molecular-genetic studies combined with a systems approach are likely to uncover additional contributing factors.The theory of nutrition diversion, an old concept, suggests that the supply of sugar to ABs activates bud release.The study by Barrera-Rojas et al. suggest that ethylene biosynthesis and sugar transport are also modulated in ABs in addition to auxin and ABA.Emerging evidence suggests that sugar transport significantly influences the release of ABs parallel to auxin transport (Beveridge et al., 2023).Similarly, the inhibition of AB growth during shade avoidance strongly correlates with the stimulation of ethylene biosynthesis (González-Grandío et al., 2013).
Our understanding of the quiescence and activation of ABs is still incomplete.However, the work of Barrera-Rojas et al. provides new insights into AB growth.The controlled modulation of ABs could have a significant impact on plant architecture and improve agronomic yields.The overexpression of miR156 down-regulates SBP15 levels, resulting in reduced IAA and ABA levels in the bud.The impact of SBP15 on GOBLET levels in ABs is much less pronounced than in rSBP plants.The decrease in IAA levels is likely to be caused by the initiation of auxin canalization, which promotes the outgrowth of ABs.

Fig. 1 .
Fig. 1.The miR156/SBP15 module controls tomato axillary bud (AB) growth.(A) Overexpression of miR156-resistant SBP (rSBP15) strongly induces AB quiescence.This quiescence is characterized by the downregulation of auxin transporters PIN9 and LAX2, and the accumulation of IAA and ABA.rSBP15 also decreases the expression of GOBLET, a leaf dissection-related gene.The down-regulation of auxin transporters may prevent the canalization of auxin, thereby leading to AB quiescence.(B)The overexpression of miR156 down-regulates SBP15 levels, resulting in reduced IAA and ABA levels in the bud.The impact of SBP15 on GOBLET levels in ABs is much less pronounced than in rSBP plants.The decrease in IAA levels is likely to be caused by the initiation of auxin canalization, which promotes the outgrowth of ABs.